Method for detection of an analyte in a fluid sample

ABSTRACT

A method for detecting an analyte in a fluid sample is disclosed. The method comprises: a) providing a measurement region and a reference region, the measurement region being provided with a receptor for binding the analyte; b) providing at least one light beam so as to travel along the measurement region and along the reference region; c) providing the fluid sample into at least the measurement region; d) detecting by means of a detector an optical pattern provided by the at least one light beam after having travelled along the measurement region and the reference region; and e) deriving a presence of the analyte in the fluid sample from the detected optical pattern, wherein prior to c) a blocking fluid is provided along the measurement region and along the reference region.

The invention relates to a method and measurement system for detectionof an analyte in a fluid sample. Furthermore, the invention relates to adisposable measurement structure.

There is an increasing need for highly sensitive methods, which arerequired to detect various types of analytes such as micro-organisms,proteins, DNA molecules, etc., and to measure their concentration in agiven fluid sample solution such as sample liquid, e.g. body fluid,milk, drinking or waste water, etc., vapour or gaseous sample. In thelast couple of years, the use of the sensors in medical diagnostics,food and water safety, security applications, animal and plant healthmonitoring, environmental monitoring, etc., is becoming increasinglyimportant. In a sensor device, the receptor layer, e.g. an antibodylayer, which is immobilized on the sensor surface, is an importantcomponent that selectively binds to/interacts with the specific analytethat is present in a given sample solution. The role of the receptorlayer becomes especially important when the specific analyte needs to bedetected in samples such as serum, blood, milk, etc., where othernon-specific components, e.g. proteins and DNA molecules, are present aswell. In recent years different coating procedures have been developedto provide/improve the specificity of receptor-analyte interactions,e.g. by preventing and/or reducing the non-specific interactions. Inclinical and food applications, usually complex samples such as serum,blood, milk, etc., in which the concentration of non-specific componentsis much higher than the concentration of the specific analytes, need tobe analyzed. An example could be detection of very low concentrations ofbiomarkers in blood or other relevant body fluids that could lead toearly disease detection diagnosis and prevention/treatment. The presenceof a high background in clinical samples can result in deterioration ofthe specificity of these sensors. A lower specificity implies further adecrease of the accuracy and sensitivity of the sensor.

The invention intends to improve an analyte detection.

In order to achieve this goal, the method according to the inventioncomprises:

a) providing a measurement region and a reference region, themeasurement region being provided with a receptor for binding theanalyte;

b) providing at least one light beam so as to travel along themeasurement region and along the reference region;

c) providing the fluid sample into at least the measurement region;

d) detecting by means of a detector an optical pattern provided by theat least one light beam after having travelled along the measurementregion and the reference region; and

e) deriving a presence of the analyte in the fluid sample from thedetected optical pattern.

The optical light beam travels across the measurement and referenceregions in various ways. It is for example possible that the beam issplit up by a divider or other splitter in a measurement beam and areference beam, respectively travelling across the measurement regionand the reference region. Alternatively, it is possible that themeasurement and reference regions together form a waveguide structurewhich allows passage of the beam in two or more propagation modes. Themeasurement and reference regions may thereby be assigned to respectiveparts of the waveguide structure, examples of which will be providedbelow. The optical radiation from the measurement and reference regionsmay then interact with each other, e.g. by means of interference,resulting in a pattern, such as an interference pattern, on a surface ofthe detector. As a result of binding of an analyte (e.g. a molecule,assembly of molecules, or molecule group, virus, bacteria, cell, etc) onthe sensor surface of the measurement region which is coated with areceptor layer, an optical behavior of the respective region will bealtered, which results in a change in a property (e.g. a phase change)of the light beam or light beam propagation mode from the respectiveregion. As a result thereof, the interference pattern will show achange, the resulting pattern being detected by the detector andanalyzed. The presence (e.g. a concentration, a change of concentration,an occurrence, binding kinetics, affinity to the receptor, etc.) of theanalyte may be derived therefrom.

The deriving a presence of the analyte in the fluid sample from thedetected optical pattern may comprise measuring a differential signalbetween the measurement and reference regions. By measuring adifferential signal, similar effects occurring in both measurement andreference regions will substantially compensate each other. At thedetector, an optical pattern results such as an interference patternfrom an interference between the beam having travelled along themeasurement region and the beam having travelled along the referenceregion. The interference pattern is detected by the detector andprocessed, such as by performing a Fourier transform (e.g. a fastFourier transform FFT) on the detected interference pattern. A value(such as a single value) may be derived from the processed data. Forexample, from the Fourier transformed interference pattern, a spatialfrequency peak is selected that relates to the interference between thetwo regions in question, and a phase of the selected spatial frequencypeak is represented by a single value. In case a plurality of regionsare used, for each relevant pair of regions, a spatial frequency peak isselected that represents an interference between that pair of regions.The phase value corresponding to each pair of channels is extracted atthe phase part of the FFT at the given spatial frequencies.

Non-specific binding, which may stem from binding to a.o. non-specificbinding sites of the receptor and/or from non-specific binding to sensorsurface, usually occurs simultaneously with the specific binding,resulting also in a change in the optical behavior of the measurementregion, thereby resulting in an additional change of the detectedpattern, which reduces the specificity of the measurement.

In order to improve a specificity of the measurement, prior to c) ablocking fluid may be provided along the measurement region and alongthe reference region. The blocking fluid may for example comprisecomponents which provide for a non-specific binding in the measurementregion, preferably without significantly changing a capability of thereceptor layer to bind the analyte, and the reference region, examplesof the blocking fluid including e.g. a serum that does not contain theanalyte, or any other fluid containing a component that provides for anon-specific binding but that does not contain the analyte. In thisembodiment, the reference channel may but does not necessarily need tobe provided with the sample. Instead, the fluid sample can be providedin the measurement region only. Thereby, a reference fluid (such as aserum, other examples provided elsewhere in this document) may beapplied in the reference channel. For clarification reasons, morespecific examples of the blocking fluid include, but are not limited to,a fluid comprising Protein A, Bovine Serum Albumine (BSA), casein, orgelatine or a combination of these. Under ideal conditions the blockingfluid would also include a non-specific receptor, which e.g. can be anantibody non-specific to the analyte of interest or an oligo (DNA/RNA)molecule/string not specific to the analyte of interest or an enzyme notspecific to the analyte of interest. Such to mimic the circumstances fornon-specific blocking in the reference and the measurement regions asclosely as possible. Ideally the only difference would be the presenceof the specific binding site in the measurement region. In other words:both the reference and measurement regions may initially be coated bythe blocking fluid, e.g. with abundant Protein A comprised in theblocking fluid, to reduce the non-specific binding to the sensor surfaceof as well the measurement region as the reference region. The blockingfluid may thereby saturate e.g. a bulk of the non-specific binding sitesat the receptor (present in the measurement region only) and/orgenerally at the sensor surface in the measurement region and/or thereference region. By the application of such a blocking fluid, both themeasurement and reference regions are initially coated with non-specificcomponents, the providing of the fluid possibly containing the analyteto be detected may mostly result in specific binding only, asnon-specific binding has already taken place to a substantial extent bythe application of the blocking fluid. The blocking fluid may be appliedbefore or after the receptor has been provided in the measurementregion. If for example the blocking fluid comprises Protein A, providingthe blocking fluid in the measurement region before the provision of thereceptor, may result in an improved orientation and anchoring of thereceptor in the measurement region. However, in case the blocking fluidis applied after having provided the receptor in the measurement region,non-specific binding sites on the receptor itself may be saturated bythe blocking fluid so as to keep open substantially only the specificbinding sites of the receptor in the measurement channel.

Furthermore, it is also possible to provide a modified receptor in thereference region, the modified receptor being modified in that itsspecific binding capabilities for binding the analyte are removed.Thereby, a similarity between the measurement region and referenceregion may be further improved so as to further reduce effects of thenon-specific binding on the measurement results.

The light beam may comprise any suitable beam, e.g. a substantiallycoherent beam, a substantially monochromatic beam, multiple wavelengthsbeam, or a beam having a spectrum substantially continuously extendingover a wavelength range (such as white light or other super continuum)etc. The beam may be in any suitable wavelength range, e.g. visible ornear infrared, infrared, ultraviolet, and may be generated by anysuitable optical source, such as a laser, a semiconductor laser diode, asuperluminescent diode, a VCSEL (vertical-activity surface-emittinglaser), a light emitting diode equipped with suitable filters such aspolarizing filters, etc. The detector may comprise a CCD (charge coupleddevice) or other suitable camera such as CMOS (complementarymetal-oxide-semiconductor), and may be formed by e.g. a line array ortwo dimensional pixel array. The processing of the detected pattern maybe performed by any suitable processing device (e.g. a microcontroller,microprocessor, embedded controller, personal computer, single boardcomputer, personal digital assistant, etc) provided with suitablesoftware, or by suitable dedicated electronics. The processing may beperformed in real time during the image capturing, allowing e.g.performance of a real-time kinetics measurement or in-line productionanalysis, or at a later moment in time. An example of a suitableprocessing is described e.g. in S. Nakadate (1988) J. Opt. Soc. Am. A 5,1258-1264.

The white light or super continuum may provide for more (accurate)information to be obtained in a nanometer domain of the analysis, e.g.at nanometer distance of the sensing surface. Other light beams may e.g.be more suitable for obtaining information at larger distances from thesensing window.

In accordance with an embodiment of the invention, the effects ofnon-specific binding on the measurement results may be reduced, as—dueto the fact that the sample fluid is brought to the measurement region(e.g. measurement channel) as well as to the reference region (e.g.reference channel), non-specific binding will occur on both channels—asopposed to the known configurations wherein the specific as well as thenon-specific binding both take place in the measurement region only. Asa result of the occurrence of the non-specific binding in themeasurement region as well as in the reference region, the effectsthereof on the pattern as detected by the detector, may at least partlycompensate each other, as a differential signal between the measurementregion and the reference region may be largely due to the effects of thespecific binding. As a result, a lower sensitivity towards non-specificbinding may occur, hence improving the sensitivity of the measurement.

In case the blocking fluid is brought into the measurement and referenceregions (e.g. channels) and the fluid sample is brought into themeasurement and reference regions, a highly accurate measurement may beprovided. By coating not only the measurement region with the blockingfluid, but also the reference region, a similar non-specific layer dueto the binding of the blocking fluid components to the non-specificbinding sites on the sensor surface will be immobilized on both themeasurement region and the reference region. In this case, thedisturbing factors that may be present during a binding event occurringin the measurement region, e.g. temperature changes, etc., may be bettercompensated between the measurement region and the reference regionbecause the (optical) layer structure of the reference region becomes asclose as possible similar to the (optical) layer structure of themeasurement region. As such, the signal caused by the temperaturechanges in the reference region may be closer to the signal caused bythe temperature changes in the measurement region, as compared to thesituation when no blocking fluid is used in the reference region.Therefore, the compensation/cancellation of the signal due to thetemperature changes may be more effective when blocking fluid isprovided not only on the measurement region, but also in the referenceregion. This may result in a more stable differential signal measuredfor the specific binding when both the measurement region and thereference region are provided with the blocking fluid compared to thesituation when only the measurement region is provided with the blockingfluid.

Another disturbing factor that could influence the stability of thesensor signal measured for the specific binding is thedesorption/detachment of the components, e.g. of the blocking fluid,which may be weakly bound, from the sensor surface of the measurementregion, but also from the antibody layer. This factor may becomerelevant especially when a flow system is used for coating of the sensorsurface and application of the analyte sample, as it occurs in theinterferometric based devices mentioned above. Providing the referenceregion with the blocking fluid may result in a comparable signal due todesorption/detachment of the blocking fluid components from the sensorsurface of the reference region, which may largely compensate/cancel asignal due to desorption/detachment of the blocking fluid componentsfrom the sensor surface of the measurement region, resulting in a morestable differential signal corresponding to the specific binding.

In an embodiment, after having applied the blocking fluid to both themeasurement and reference region, the sample fluid is applied to boththe measurement region and the reference region. If the sample fluidwill be provided not only into the measurement channel, but also intothe reference channel, which both were previously coated with theblocking fluid, then next to a more stable differential signal, asillustrated above, a reduction/compensation of a bulk effect between thesample fluid and the blocking fluid may be provided. This may result ina more accurate measurement, for example an accurate estimation of aninitial slope of a binding curve, which is used to derive the presenceof the analyte during the very first minutes after application of thesample fluid. The term bulk effect is defined as the signal (i.e. thedifference in the interference pattern) that results when instead of asame fluid in the measurement and reference region, a fluid is appliedinto the measurement region and a different fluid is applied into thereference region, e.g. blocking fluid and sample fluid. Furthermore,when the sample fluid is provided into both the measurement region andthe reference region, the additional non-specific binding that may becaused by sample fluid components other than specific analyte will becompensated between the measurement region and the reference region,contributing therefore to a more accurate differential signalcorresponding to the specific binding. In case the blocking fluid isapplied after having provided the receptor in the measurement region,non-specific binding sites on the receptor itself may be saturated bythe blocking fluid so as to keep open substantially only the specificbinding sites of the receptor in the measurement channel.

In an embodiment, a differential measurement is performed. As explainedabove, an interference pattern is obtained from the light beam havingtravelled along the measurement region and along the reference region.By measuring a differential signal, similar effects occurring in bothmeasurement and reference region will substantially compensate eachother. In a further embodiment, a change over time of the differentialsignal is measured, thereby measuring. the changes over time in theinterference pattern: in other words, in an embodiment, the opticalpattern is detected in accordance with d) before and after providing thesample fluid in the measurement and/or reference regions, and e)comprises deriving a presence of the analyte in the fluid sample from achange in the detected optical pattern before and after providing thesample fluid in the measurement and/or reference regions. As similareffects in the measurement and reference region substantially compensateeach other, and as in a preferred embodiment the blocking fluid isbrought into both the measurement and the reference region, and thesample fluid is also brought in both the measurement and the referenceregion, similar conditions are provided in the measurement and referenceregions, and as a result any changes observed in the interferencepattern—should (almost) entirely be due to the (built up) presence ofanalyte of interest in the measurement region. Whereas the analyte ofinterest does not specifically bind in the reference region, and whereasthe presence of the specifically bound analyte in the measurement regioncauses a phase change in the measurement region which does not occur inthe reference region, this difference in phase change between themeasurement and the reference region causes a change in the interferencepattern, which can be analyzed over time and which change has a directrelationship with the concentration of the analyte of interest in themeasurement region. As a result of the similar conditions in themeasurement and reference regions (in particular when the blocking fluidis brought in the measurement and reference region and the sample fluidis brought in the measurement and reference region as well), disturbingfactors will compensate each other to a large extent, so that a changein the interference pattern will result almost entirely from a buildupof the analyte in the measurement region. An aim of the underlyingmethod is thus to impose a difference in the optical behaviour betweenthe measurement and the reference region, whereby this difference shouldideally be entirely due to specific binding of the analyte of interestin the measurement region.

In somewhat more general wording, the above principle may be describedas follows: In an embodiment of the invention, the method furthercomprises detecting before c) by means of the detector a referenceoptical pattern provided by the at least one light beam after havingtravelled along the measurement region and the reference region,

wherein d) is performed at least once during or after providing of thefluid sample into at least the measurement region,

and wherein e) comprises:

comparing a characteristic of the reference optical pattern with thecharacteristic of the optical pattern detected in d), and obtaining thepresence of the analyte therefrom.

Whereas the analyte of interest does not specifically bind in thereference region, and whereas the presence of the specifically boundanalyte in the measurement region causes a phase change in themeasurement region which does not occur in the reference region, thisdifference in phase change between the measurement and the referenceregion causes a change in the optical pattern (for example theinterference pattern), which can be analyzed over time and compared tothe (reference) optical pattern obtained before applying the fluidsample, and which change may have a direct relationship with theconcentration of the analyte of interest in the measurement region. As aresult of the relatively similar conditions in the measurement andreference regions (in particular but not exclusively when the blockingfluid is brought in the measurement and reference region and the samplefluid is brought in the measurement and reference region as well),disturbing factors will compensate each other to a large extent, so thata change in the interference pattern will result almost entirely from abuildup of the analyte in the measurement region. The further effects asdescribed in the above paragraph may apply to this embodiment likewise.It is noted that the reference optical pattern may in this document alsobe referred to as the optical pattern or the interference patterndetected before application of the fluid sample into the measurementregion (and possibly the reference region) or any other similar wording.The term reference optical pattern is thus to be understood as anoptical pattern detected before the providing of the sample fluid in c).

In order to achieve accurate results while analyzing the patternsquickly, in an embodiment, the characteristic of the optical pattern andthe reference optical pattern comprises a phase of a frequency componentin a spatial frequency spectrum (e.g. obtained by means of a fastfourier transform) of the optical pattern, the frequency component froman interference between the at least one light beam having travelledalong the measurement region and having travelled along the referenceregion.

Another disturbing factor that often limits the sensitivity in a sensordevice is the presence of drift, e.g. due to temperature changes thatoccur e.g. when sample solutions that need to be analyzed are brought tothe sensor surface. Drift can also be caused by temperature changes ofthe environment, heat exchange during a binding event, etc. Because thesignal due to the drift occurs simultaneously with the signal due to thespecific binding, during the time frame of a binding event it ispractically impossible to discriminate between specific binding signalsand drift signal. This may cause a further decrease of the specificityand sensitivity of the sensor.

In a further embodiment of the method, a second reference region isprovided,

wherein d) further comprises measuring a deviation between the referenceregion and the second reference region, and

wherein e) further comprises estimating a disturbance from the deviationmeasured in d) between the reference region and the second referenceregion, and correcting the information concerning the presence of theanalyte for the estimated disturbance.

Making use of this concept, disturbances, such as a drift (e.g. due totemperature effects), an effect of non-specific binding, or othereffects may be at least partially be corrected for by using themeasurement between the reference region and the second reference regionto obtain information that may be applied to correct for thisdisturbance.

As an example, an effect of drift may at least partially be compensatedby measuring a drift between the reference region and the secondreference region, estimating the drift between the measurement regionand the reference region from the measured drift between the referenceregion and the second reference region.

In order to provide an accurate estimation, before provision of thefluid sample, i.e. before c), a first drift may be measured between themeasurement region and the reference region, and a second drift may bemeasured between the reference region and the second reference region.Thereby, a drift relation can be determined between the first and seconddrifts. These drift measurements may be performed with a reference fluidin one or more of the regions, preferably in each one of the regions, soas to obtain similar conditions in each of the regions. Hereby, thereference fluid can actually be chosen to mimic the sample fluid asclosely as possible, such to ideally have the only difference betweenthe reference fluid and the sample fluid stemming from the potentialpresence of the analyte in the sample fluid. After having performed thedrift measurements, the sample is provided in at least the measurementregion. A measurement of the measurement region in respect of thereference region is performed. Furthermore, a measurement of thereference region in respect of the second reference region is performed.A drift that occurs during the measurement between the measurementregion and the reference region may now be estimated from the determineddrift relation, and a measurement between the reference region and thesecond reference region (which expresses the drift occurring during themeasurements between the reference region and the second referenceregion). The measurement between the measurement region and thereference region (which should ideally only express the binding of theanalyte) can now be corrected for the estimated drift between theseregions, which may reduce an adverse effect of drift on the measurementaccuracy. In other words, the effects of drift in the specific bindingsignal may be reduced, as the drift signal measured between thereference region and the second reference region may be used todetermine or estimate the drift that occurs between the measurementregion and reference region. This could be achieved by e.g. determiningthe relation between measured signals for each pair of regions prior toapplication of the fluid sample containing the analyte. Examples ofreference fluids include, but are not limited to, serum not containingthe analyte, solutions containing Protein A or BSA or can even consistof pure buffer such as PBS (phosphate buffered saline). Forclarification: the actual sample—possibly containing the analyte—will beintroduced at the (first) reference region and the measurement region,but preferably not in the second reference region. This latter regionwill preferably be exposed to the reference fluid, whereby the referencefluid is brought simultaneously or sequentially to the second referenceregion during the time when the sample is introduced at the (first)reference region and the measurement region.

The above concept of the provision of a second reference region may berepeated by addition of a third reference region, etc so as to be ableto take account of two or more disturbances. In an embodiment, a thirdreference region is provided, wherein d) further comprises measuring adeviation between the second reference region and the third referenceregion, and

wherein e) further comprises estimating a further disturbance from thedeviation measured in d) between the second reference region and thethird reference region, and correcting the deviation between thereference region and the second reference region for the estimateddisturbance between the measurement region and the reference region.

As an example, during a measurement, the second and third referenceregions are provided with a reference fluid, while the measurementregion and the reference region are provided with the sample. Ameasurement of the deviation between the second and third referenceregions provides an indication of the effect of drift. A measurement ofthe deviation between the (first) reference region and the secondreference region provides a combination of effects of drift and effectsof non-specific binding (as the sample is in the reference region only).The measurement between the measurement region and the reference regioncan now be corrected for an estimation of the drift (obtained from themeasurement between the second and third reference channels, possibly incombination with a determined drift relation as described above) and forthe effects of non-specific binding.

For clarification purposes: in such embodiments, the sample potentiallycontaining the analyte will preferably not be introduced in referenceregions two and three. These reference regions are preferably exposed tothe reference fluid. The effects of drift in the specific binding signalmay be reduced, as the drift signal measured between the secondreference region and the third reference region may be used to determineor estimate the drift that occurs between the measurement region and the(first) reference region. Also, the contribution of a possiblenon-specific binding of the analyte in the (first) reference region tothe specific binding signal may be reduced by correcting/reducing thedrift signal between the (first) reference region and the secondreference region.

This could be achieved e.g. by determining the relation between measuredsignals for each pair of regions prior to application of the fluidsample containing the analyte.

In a further embodiment, e) comprises determining an initial slope of ameasurement curve and deriving the presence of the analyte from thedetermined initial slope. Thereby, the initial slope may be used toextrapolate the concentration of the analyte.

Usually, binding of the analyte to the receptor is slow, and may take upto several hours until a saturation of the binding has been achieved.Determining the initial slope of the measurement curve, such as theanalyte binding curve between the measurement region and referenceregion (as derived from the detected pattern) may allow to derive apresence and/or concentration of the analyte there from within arelatively short time frame, such as in several minutes. Hence,saturation of the measurement curve may not be required to quicklydetermine the concentration of an analyte, whereas a steepness of theinitial slope directly relates to the concentration. This is explainedfurther below.

In a still further embodiment, the method comprises the further stepsof:

removing at least part of the analyte from the receptor layer by aremoval process, the optical pattern being detected before and after theremoval. Thereby, an accuracy can be further enhanced, as a measurementis performed before and after removal of the analyte, which improves anability to discriminate an effect of binding of the analyte fromnon-specific binding, drift, and other factors, as a signal changeobtained due to the removal may be due to the amount of analyteparticles detached by the removal process. Any suitable removal processmay be applied, e.g. providing a dedicated solution, such as an HClacidic solution or an ionic gradient solution or a solution containing acompetitor molecule. In such embodiment, the reference fluid may beapplied along the reference region and the removal process may furtherbe performed along the reference region and the measurement region, e.g.simultaneously. Thereby, a possible removal of non-specific componentsfrom the measurement region during the removal process may becompensated by a removal of non-specific components from the referenceregion.

In another configuration of such an embodiment, it is further possiblethat the reference fluid is further applied along the second referenceregion, that the removal process is further performed along the secondreference region, and that e) comprises deriving a drift between themeasurement region and the reference region from a drift measuredbetween the reference region and the second reference region, andcorrecting the information concerning the presence of the analyte forthe derived drift between the measurement region and the referenceregion. Thereby, in analogy with the above described embodiment whereina second reference region is applied, a differential signal between thereference region and the second reference region (which may be caused bytemperature changes and other disturbing factors) may further be used tocorrect for a drift signal between the measurement region and thereference region.

In a yet further embodiment, the light beam comprises at least twospectrally distinct wavelength ranges or polarization ranges, thedetection being performed for each of the wavelength or polarizationranges. The ranges may e.g. each comprise a specific wavelength and/orpolarization. For different wavelengths and/or different polarizations,a different sensitivity may be obtained for various binding events, asthe various components that result in binding (e.g. viruses, proteins,protein assemblies or protein groups, bacteria, cells) may havedifferent dimensions. The different sensitivities may be applied—whenusing multiple wavelengths and/or polarizations, to determine an effectof different contributions (specific binding, non-specific binding, etc)from the different responses at the different wavelengths and/orpolarizations. Making use of these differences in sensitivity, in anembodiment, three distinct wavelength ranges are comprised in the lightbeam, and e) comprises determining analyte binding, non-specific bindingand bulk refractive index from the detected optical patterns for each ofthe wavelengths.

In an embodiment, the method further comprises:

detecting a scattering of light from the measurement and referenceregions and combining the detected light scattering and/or localintensity distributions with the detected optical pattern in order toderive the presence of the analyte in step e). Thereby, additionalinformation regarding the specific binding events in the measurementregion may be obtained from the scattered signal and spatial intensitydistribution, allowing a further improvement in measurement accuracy andsensitivity.

In accordance with a further embodiment of the invention, a compensationof the bulk effect may be provided. Thereto. the bulk effect between themeasurement region and the reference region is measured: the samplefluid is brought into the measurement region and a reference fluid isbought into the reference region, an interference pattern between themeasurement and reference regions being detected and stored in a memory(for example by storing the pattern or by storing relevant informationobtained from a fast fourier transform of the interference pattern, suchas a phase value of a frequency peak in the fast fourier transformspectrum). When a measurement is performed, whereby the sample is in themeasurement region and the reference fluid is in the reference region,the stored information that represents the bulk effect may be applied tocorrect for the bulk effect, i.e. for the contribution of the differentfluids to the interference pattern.

As an example: in the reference region, a PBS buffer with an RNA string(probe) is brought. In the measurement region, a sample (such as aserum) is brought that contains the RNA string (probe) and possibly acomplementary string whose presence is to be detected. Antibodies areprovided in the measurement and reference region. RNA strings and ifpresent, the complementary strings, are bound to the antibodies by meansof a tag. The stored value(s) that represent an effect of the differentfluids in measurement and reference regions, may be applied to correct ameasured interference pattern, so as to substantially remove an effectof the different fluids on the interference pattern and measurement soas to more accurately measure a contribution of binding of the analyte.In both the measurement and reference region, the RNA probes bind withthe tag to the antibodies, however only in the measurement channel thecomplementary RNA/DNA (i.e. the analyte) binds to the probe, which probehas a tag. This tag is subsequently bound to the antibody on the chipsurface.

In the above method it is also possibly that the sample fluid, such as aserum, is brought into both the measurement and reference region so asto keep the circumstances in both regions as close as possible. In thatcase, the probe in the reference region should be a dummy so as to avoidany binding of the analyte in the reference region. Both measurementmethods may also be applied simultaneously (whereby two referenceregions are required as also described below) so as to obtain moreinformation and consequently a higher accuracy.

According to a further aspect of the invention, a measurement system isprovided for detecting an analyte in a fluid sample, comprising:

a measurement region and a reference region, the measurement regionbeing provided with a receptor for binding the analyte;

a light source for generating at least one light beam so as to travelalong the measurement region and along the reference region;

a fluid supply for providing the reference fluids and for the fluidsample into the measurement region and the reference region;

a detector for detecting an optical pattern provided by the at least onebeam after having travelled along the measurement region and thereference region; and

a data processing device for deriving a presence of the analyte in thefluid sample from the detected optical pattern.

With the measurement system, the same or similar advantages may beachieved as with the method according to the invention. Furthermore, thesame or similar embodiments may be provided, each providing same orsimilar advantages as with the method according to the invention.

In an embodiment, at least the measurement region and the referenceregion are provided on a planar structure (also referred to as chipstructure), the measurement system comprising holding means forreplaceably holding the chip structure. Thereby, a versatile measurementsystem may be created: measurements may be performed for differentanalytes by making use of corresponding chip structures which are eachprovided with a suitable receptor for the specific analyte to bemeasured. A variety of samples can be analyzed with a respective chipstructure by providing each of the samples on the respective chipstructure and placing the chip structures (e.g. one after the other) inthe measurement system. Cross contamination of samples may be preventedin that the different samples are each applied to a different chip.Different samples can also be applied to different (measurement) partson one and the same chip.

The planar structures (also referred to as “chips”) may be in partmanufactured in a semiconductor material patterning and etching process,thereby allowing to supply them at a reasonable cost. Alternatively,other (optically) suitable materials may be applied. In order to detectvarious analytes, different receptors may be provided on the respectivemeasurement regions of such chips. The comparably low cost furtherallows one time use, thereby facilitating handling and obviatingregeneration/cleaning after each measurement.

The fluid supply may be provided with a reservoir, e.g. a microreservoir for holding a (small) amount of the fluid to be analyzed, thefluid then being provided to the measurement and/or referenceregion/channel by capillary force e.g. through a (micro-)fluidic systemthat forms part of the chip and which comprises (micro-)fluidic channelsthat specifically address/are coupled to one reference region/channel orone measurement region/channel, a (micro-) fluidic pump, gas pressure,etc. A fluid can also be flowed continuously over one or more specificparts of the chip. The chip can be either disposable or enable re-usageas explained below. The feature that of the measurement region and thereference region being provided on a chip structure, the measurementsystem comprising holding means for replaceably holding the chipstructure, can not only be applied in the measurement system accordingto the invention, but also in any other interferometer based measurementsystem. Hence, such measurement system could also be described as:

a measurement system for detecting an analyte in a fluid sample,comprising:

a measurement region and a reference region, the measurement regionbeing provided with a receptor for binding the analyte;

a light source for generating at least one light beam so as to travelalong the measurement region and along the reference region;

a fluid supply for providing the fluid sample into at least themeasurement region;

a detector for detecting an optical pattern provided by the at least onebeam after having travelled along the measurement region and thereference region; and

a data processing device for deriving a presence of the analyte in thefluid sample from the detected optical pattern, wherein

at least the measurement region and the reference region are provided ona chip structure, the measurement system comprising holding means forreplaceably holding the chip structure.

The fluid supply may also be connected to or comprised in the(replaceable) chip structure, thereby being replaceable (with the chip)at least in part, so as to e.g. prevent a next sample to be contaminatedby a remainder of a previous sample in the fluid supply. The reservoirof the fluid supply may be connected to or comprised in the chip so thateach chip has its own, however a separate reservoir may be provided asan alternative.

The chip structure and the fluid supply may be held by a holder and soas to align the fluid supply to at least the measurement region by theholder.

In the above and other embodiments of the present invention, a methodand measurement system are provided for highly specific and sensitiveanalyte detection in fluid sample solutions, e.g. liquids such asbody/animal/plant fluid (serum, plasma, blood, sputum, etc.), milk,drinking or waste water, etc., vapours or gasses such as air, which e.g.can be pre-treated and diluted into a liquid, e.g. PBS buffer. Analytespresent in the gas sample may in this way be solved in the liquid whichmay thereupon be analyzed.

Gasses could also be detected using gas absorbent layers that arespecific towards a given gas component, e.g. CO2, toxic gasses, etc.

Further advantages, embodiments and effects of the invention will becomeclear from the appended drawing and corresponding description, in whichnon-limiting embodiments of the invention are depicted, in which:

FIGS. 1A and B provide a general schematic view of an interferometricbased sensor and analyte binding taking place therein;

FIGS. 2A, B, C,D and E provide a schematic representation of analytebinding in different measurement schemes in order to illustrate variousembodiments of the invention;

FIG. 3 provides a schematic representation of a Young interferometerbased sensor in which various embodiments of the invention may beapplied;

FIG. 4 provides a schematic representation of a Mach-Zehnderinterferometer based sensor in which various embodiments of theinvention may be applied;

FIG. 5 provides a schematic representation of a Multi-Mode interferencebased sensor in which various embodiments of the invention may beapplied;

FIGS. 6A, B, C and D provide a schematic representation of analytebinding in different measurement schemes in order to illustrate variousembodiments of the invention;

FIG. 7 provides a schematic representation of analyte binding toillustrate embodiments of the invention;

FIG. 8 provides a schematic representation of an interferometric sensingconfiguration in order to illustrate various embodiments of theinvention;

FIG. 9 provides a schematic representation of a measurement system inaccordance with an embodiment of the invention; FIGS. 10A and B depictsembodiments of a lab-on-a-chip system to be applied in embodiments ofthe invention;

FIG. 11 provides a schematic representation of a portable detector inaccordance with an embodiment of the invention and

FIGS. 12A and B depict a time diagram of a detecting of an analyte inaccordance with embodiments of the invention.

ESTIMATION/REDUCTION OF NONSPECIFIC BINDING

FIG. 1A depicts a top view of a general schematic of an interferometricbased sensor. In an interferometric based sensor, light beam from a(monochromatic) light source LSO, e.g. a laser, is usually coupled to anoptical (channel) waveguide structure WGS. In a waveguide structure WGS,usually consisting of three layers, i.e. substrate SUB, core COR andcover COV layer (see the side view of the waveguide structure WGSdepicted in FIG. 1B), guiding of the light is performed due toappropriate refractive index contrast between the core layer and thecladding (substrate SUB and cover COV layers indicated in FIG. 1B). Ahigher refractive index of the core layer allows total internalreflection of the light at the core-cladding interface, in that waymaking possible propagation of the light through the (slab) waveguide.

On top of the waveguide structure a number of sensing regions, e.g. two,can be implemented, e.g. by locally removing the top cover layer COV;one of them can play the role of the measurement region MRG and theother one can be used as the reference region RRG. Light beamspropagating through the measurement MRG and reference RRG regionsinterfere with each other, e.g. on a screen (in this example a surfaceof an optical detector DET), generating an interference pattern.Measurement region is usually coated with a receptor REC such asantibody to enable specific detection of analytes ANA that are presentin a given solution that is flowed through the measurement region via afluidic system. Referring to FIG. 1B, specific analyte ANA binding tothe antibody-coated waveguide surface in the measurement region, whichis probed by the evanescent field of the guided modes MOD, causes acorresponding phase change that is measured as a change in theinterference pattern. Analysis of the interference pattern can yieldinformation on the amount of the analyte bound on the measurementregion. This analysis of interference pattern(s) can consist ofcomparing interference patterns before, during and after providing thesample that may contain the analyte(s) of interest to (specific regionsof) the surface of the optical waveguide structure. Variousconfigurations of interferometric based devices have been described e.g.in: C. Stamm et al. (1993) Sensors and Actuators B 11, 177-181; R. G.Heideman et al. (1993) Sensors and Actuators B 10, 209-217; A.Brandenburg et al. (1994) Applied Optics 33(25), 5941-5947; H. Helmerset al. (1996), Applied Optics 35(4), 676-680; A. Ymeti et al. (2003)Applied Optics 42, 5649-5660; G. H. Cross et al (2003) Biosensors andBioelectronics 19(4), 383-390.

In a (bio-)sensor device having multiple sensing regions, the surface ofone of the sensing regions can be first coated with a receptor layer(measurement region). In this document, the term receptor may beunderstood as a substance that specifically binds the analyte. The termanalyte may refer to e.g. a chemical or biological component (such asbut not limited to a micro organism, protein, peptide, DNA/RNA, orcombinations thereof). In a (bio-)sensor device, the receptor layer,e.g. an antibody layer, a DNA/RNA fragment that is complementary to thespecific analyte, an enzyme or other specifically analyte bindingsubstance, which is immobilized at the sensor surface, is used toselectively bind/interact with the specific analyte particles that arepresent in a given sample solution that needs to be analyzed. Anotherexample is CO2 (gas) binding at the receptor layer. The function of thereceptor layer is especially important when the specific analyte needsto be detected in very complex samples such as serum, blood, milk, etc.,where other non-specific components, e.g. proteins, micro-organisms(such as viruses, bacteria, yeasts etc.), DNA molecules, mineral ions,etc., are present as well. Depending on the application, configurationand other circumstances, it may be desirable that the receptor layer isstable, does not have or has minimal non-specific binding sites, can beimmobilized reproducibly and has high density of active receptors.

Immobilization of the receptor layer at the measurement region can beperformed using different techniques that depend on the chip material,e.g. for a chip based on Silicon (Si) one can use binding to Protein Acoated sensor surface. A Protein A coated sensor surface can be used topromote the binding and enhance proper orientation of the receptor forfurther analyte binding. Furthermore, coating the sensor surface withProtein A may result in reduction of non-specific binding to the sensorsurface. Protein A is given as an example. Other proteins or substancescan exhibit the same or similar functionality as Protein A: beingforming a cover layer at the Si surface, thereby reducing non-specificbinding to this surface and acting as proper anchor point for thereceptor such as antibodies, in order to bind and orientate the receptorin a desired way. Other techniques for immobilization of the receptorlayer could be used as well, e.g. physical adsorption on the sensorsurface, which is based a.o. on hydrophobic interactions and hydrogenbonds, or covalent coupling, e.g. to a silanized sensor surface.

Whereas the measurement region may be coated with a specific receptor,an additional second region—also referred to as reference region—may becoated only with Protein A or another protein or molecule that exhibitssimilar functionality as Protein A. This is described above as blockingfluid. The body fluid sample, e.g. serum containing a specific analytesuch as a biomarker, can be applied (simultaneously) in both regions, asschematically illustrated in FIG. 2.A. Coating the sensor surface of thereference region RRG with Protein A may also contribute to the reductionof the non-specific binding, in this case of the serum components, inanalogy with the measurement region MRG. Compared to the known measuringapproach in which usually the sample is applied only in the measurementregion and therefore it is not possible to differentiate between thesensor signal caused by the specific binding of the analyte to thereceptor layer immobilized on the sensor surface and the non-specificsignal caused by the binding of other components in the sample solutionto the sensor surface, this scheme provides the advantageous effect thatthe non-specific binding occurring in the reference region, which isalso reduced by coating its sensor surface with Protein A in similar wayas the measurement region, can largely compensate the non-specificbinding that occurs simultaneously in the measurement region. Thereforethe differential signal measured between the measurement region and thereference region is largely caused by the specific binding of theanalyte onto the receptor layer in the measurement region, considering acomparable non-specific binding of other components in the sample to thesensor surface in both these regions.

A further embodiment is illustrated with reference to FIG. 2B. In afurther application of this measuring scheme, both measurement regionMRG and reference region RRG can be first coated with the blockingfluid, e.g. to reduce non-specific binding to the sensor surface, inthis case in both the measurement region and the reference region, thenwith ‘clean’ serum sample (serum without specific analyte to bemeasured) or other (post-)blocking agents/solutions, consisting of oneor a combination (simultaneous or sequential) of reference fluid(s),which are used to block non-specific binding sites. Commonly usedblocking agents/solutions include, but without limitation, BSA (bovineserum albumin), serum, non-fat dry milk, casein, gelatin in PBS, etc. Inthis way, non-specific binding on the sensor surface and/or to thenon-specific binding sites of the receptor may even further be reduced.Next, the body fluid sample, e.g. serum containing specific analyte, canbe applied in both regions. In this configuration, because bothmeasurement and reference regions were initially fully coated withnon-specific components being present in the ‘clean’ serum sample,addition of serum containing specific analyte may result mostly insensor signal caused by the binding of the specific analyte to theantibody layer immobilized in the measurement region, while additionalnon-specific signal caused by the binding of other components in thesample is expected to be negligible or much lower than specific bindingbecause the bulk of the non-specific binding regions/sites are alreadyoccupied/blocked. As such in this measuring scheme a lower non-specificsignal, which is further compensated between the measurement region andthe reference region, may therefore contribute in a more accurate signalcorresponding to the specific binding.

A further embodiment is illustrated with reference to FIG. 2C. In afurther measuring scheme, an additional second reference region RRG 2pre-coated e.g. with Protein A may be further coated with ‘clean’ serumsample. Coating with Protein A here may have a similar purpose as in thecase of the measurement region MRG and reference region RRG, such as toreduce the non-specific binding to the sensor surface and/or to properorientate the receptor molecules. The additional exposure to clean serummay even further reduce any resulting non-specific binding in as wellthe reference and the measurement regions. The differential signal thatmay result between the reference region RRG and the second referenceregion RRG 2 is largely due to temperature differences between theseregions, resulting in the so-called drift. Other factors may includedrift in the alignment of the optical set-up. The temperaturedifferences can be caused by temperature changes of the environment,e.g. draught. A difference in the temperature of the sample solutions,which are flowed in these regions, may also result in a temperaturedifference between them. Furthermore, a temperature difference can occure.g. due to the binding event taking place in the measurement regionwhere heat exchange with the surrounding may occur. Because the signaldue to the drift in the measurement region occurs simultaneously withthe signal due to the specific binding, during the time frame of abinding event it is practically impossible to discriminate between thesignal due to specific binding and the signal due to the drift. In thismeasuring scheme, the drift signal measured between the reference regionand the second reference region may be used to correct/estimate thedrift signal that occurs simultaneously between the measurement regionand the reference region in addition to the specific signal in themeasurement region. This could be achieved e.g. by determining therelation between the signals for each pair of sensing regions prior toapplication of the sample solution containing the specific analyte.Correction/reduction of the drift signal in this measuring scheme maytherefore result in a further improvement of the accuracy of the signalmeasured for the specific binding.

The drift correction can be applied in a (bio-)sensor device that has atleast three sensing (one measurement and two reference) regions. Thiscorrection could be possible if the differential signals between themeasurement region and two reference regions are acquired, e.g.simultaneously or sequentially. It is noted that the sample—possiblycontaining the analyte—will preferably not be brought into contact withthe second reference region, whereas this sample is preferably broughtinto contact with the first reference region and with the measurementregion(s).

A further embodiment is illustrated with reference to FIG. 2D. In afurther measuring scheme, an additional third reference region, RRG 3which is pre-coated with the blocking fluid (e.g. with Protein A), maybe further coated with ‘clean’ serum sample. Coating with Protein A herehas the same purpose as in the case of the measurement region, referenceregion and second reference region, namely to reduce the non-specificbinding to the sensor surface of these regions.

The differential signal that may result between the second referenceregion RRG 2 and the third reference region RRG 3 is mostly due totemperature differences between these regions and other disturbingfactors, resulting in the so-called drift, whereas the differentialsignal between the reference region and the second reference region isdue to temperature differences between these regions and otherdisturbing factors resulting in drift signal as well as somenon-specific binding of the analyte at the sensor surface of thereference region.

The differential signal that may result between the measurement regionand the reference region is due to the specific binding of the analyteat the sensor surface of the measurement region, drift signal betweenthe measurement region and the reference region as well as thenon-specific binding of the analyte at the sensor surface of thereference region. Because the signal due to the drift between themeasurement region and the reference region occurs simultaneously withthe signal due to the specific binding in the measurement region as wellas the non-specific binding of the analyte in the reference region,during the time frame of a binding event it is practically impossible todiscriminate between the sensor signal due to specific binding in themeasuring region, the non-specific binding of the analyte in thereference region and the signal due to the drift between the measurementregion and the reference region. In this measuring scheme, the driftsignal measured between the second reference region and the thirdreference region may be used to correct/estimate the drift signal thatoccurs simultaneously between the reference region and the secondreference region as well as the drift signal that occurs between themeasurement region and the reference region. This could be achieved e.g.by determining the relation between the signals for each pair of regionsprior to application of the sample solution containing the specificanalyte. By correcting/reducing the drift signal between the referenceregion and the second reference region, the non-specific binding of theanalyte in the reference region can be estimated. Furthermore, bycorrecting the drift signal between the measurement region and thereference region and estimating the non-specific binding of the analytein the reference region, this measuring scheme may result in even afurther improvement of the accuracy of the signal measured for thespecific binding of the analyte in the measurement region.

This scheme could be applied in a (bio-)sensor device that has at leastfour sensing (one measurement and three reference) regions and if theinterference signals between the measurement region and three referenceregions are acquired, e.g. simultaneously or sequentially.

Thus, in the above embodiment, the sample, potentially containing theanalyte to be detected, will preferably not be brought into contact withthe second and the third reference regions, whereas this sample willpreferably be exposed to the first reference region and the measurementregion(s).

In a further measuring scheme, an additional fourth reference channel,e.g. in a multichannel YI based sensor or any other interferometricconfiguration having at least five sensing regions/channels (onemeasurement and four reference regions/channels), is (pre-)coated withthe blocking/reference fluid, e.g. with Protein A, having the samepurpose as in the case of the measurement channel, (first) referencechannel, second reference channel and third reference channel, namely toreduce the non-specific binding to the sensor surface of these channels.In the fourth reference region sample not containing the analyte can beflowed (see schematic in FIG. 2E).

The differential signal that may result between the second referencechannel and the third reference channel is mostly due to temperaturedifferences between these channels and other disturbing factors,resulting in the so-called drift, whereas the differential signalbetween the third reference channel and the fourth reference channel isdue to temperature differences between the third reference channel andthe fourth reference channel and the bulk signal between the sample (notcontaining the analyte) flowed in the fourth reference channel andblocking/reference fluid flowed in the third reference channel.Furthermore, the differential signal between the (first) referencechannel and the second reference channel is due to temperaturedifferences between these channels and other disturbing factorsresulting in drift signal, the bulk signal between the sample(containing the analyte) flowed in the (first) reference channel and theblocking/reference fluid flowed in the third reference channel as wellas some non-specific binding of the analyte at the sensor surface of the(first) reference channel. Finally, the differential signal that mayresult between the measurement channel and the (first) reference channelis, as in the previous measuring scheme, due to the specific binding ofthe analyte at the sensor surface of the measurement channel, driftsignal between the measurement channel and the (first) reference channelas well as the non-specific binding of the analyte at the sensor surfaceof the (first) reference channel.

In this measuring scheme, the drift signal measured between the secondreference channel and the third reference channel may be used tocorrect/estimate the drift signal that occurs (simultaneously) betweenthe third reference channel and the fourth reference channel, (first)reference channel and the second reference channel as well as the driftsignal that occurs between the measurement channel and the (first)reference channel. This could be achieved e.g. by determining therelation between the signals for each pair of channels prior toapplication of the sample solution containing the specific analyte. Bycorrecting the drift between the third reference channel and the fourthreference channel, the bulk signal between the sample (not containingthe analyte) flowed in the fourth reference channel andblocking/reference fluid flowed in the third reference channel can beestimated, which is comparable to the bulk signal between the sampleflowed in the (first) reference channel and the blocking/reference fluidflowed in the second reference channel. Furthermore, by correcting thedrift signal between the (first) reference channel and the secondreference channel and the bulk signal between the sample flowed in the(first) reference channel and the blocking/reference fluid flowed in thesecond reference channel, the non-specific binding of the analyte in the(first) reference channel can be estimated. Finally, by correcting thedrift signal between the measurement channel and the (first) referencechannel and estimating the non-specific binding of the analyte in the(first) reference channel, this measuring scheme may result in even afurther improvement of the accuracy of the signal measured for thespecific binding of the analyte in the measurement channel. This schemecould be applied if the interference signals between the measurementchannel and four reference channels can be obtained, eithersimultaneously or sequentially.

An alternative measuring scheme can be applied when blocking/referencefluid containing the analyte, preferably having the same concentrationas in the sample solution, will be flowed in the fourth referencechannel instead of the sample not containing the analyte, as describedabove. In this scheme similar results with the above scheme can beobtained.

In all above schemes, reference channels can be interchanged with eachother, e.g. the sample solution containing the analyte can be flowed inthe measurement channel and either the first, second, third or fourthreference channel.

A Young interferometer (YI) based sensor has been described in: A.Brandenburg et al. (1994) Applied Optics 33(25), 5941-5947; H. Helmerset al. (1996), Applied Optics 35(4), 676-680; A. Brandenburg (1997)Sensors and Actuators B 38-39, 266-271; A Ymeti et al. (2003) AppliedOptics 42, 5649-5660; G. H. Cross et al (2003) Biosensors andBioelectronics 19(4), 383-390. In a YI based sensor, light beam from a(e.g. monochromatic) light source LSO, e.g. a laser, is usually coupledinto an input (channel) waveguide structure OPC, and is usually split.by a beam splitter such as a network of Y-junctions (as schematicallyillustrated in FIG. 3), MMI coupler, star coupler, etc, into at leasttwo beams, which propagate through respective measurement channels MCHand reference channels RCH 1, RCH 2, RCH 3 of the waveguide structure,the measurement channels and reference channels forming examples ofmeasurement regions and reference regions respectively. The outputdivergent beams overlap with one another and the final interferencepattern can be a superposition of individual interference patterns, eachof them representing the overlap of the divergent beams of a specificchannel pair, which can have a unique distance between its channels,e.g. in a configuration with more than two channels. The interferencepattern can be recorded by a detector, in this example provided by a CCD(charged coupled device) camera, which is placed at a given distancefrom the endface of the waveguide structure. The CCD is coupled to acomputer system to process the data related to the detected interferencepattern. The computer applies an analysis algorithm, e.g. based on a FFT(fast Fourier transformation), to this data, from which the phaseinformation for each pair of channels can be (simultaneously orsequentially) determined.

FIG. 4 schematically depicts a Mach-Zehnder interferometer based sensorconfiguration. In a Mach-Zehnder interferometer (MZI) based sensor, anexample of which being disclosed in E. F. Schipper et al. (1997) Sensorsand Actuators B 40, 147-153, light beam from a (e.g. monochromatic)light source LSO, e.g. a laser, is split, e.g. using a Y-junction, so asto propagate into a measurement channel MCH and a reference channel RCHwhich form examples of a measurement region and reference regionrespectively, and after propagating through the waveguide structure OPC,light beams are combined, e.g. using again a Y-junction. The out-coupledlight intensity is recorded by a detector, in this example a photodiodePHD.

In a sensor configuration based on a YI or MZI or any otherinterferometer configuration having a measurement channel and areference channel, each output channel can be provided with a sensingwindow to allow application of fluid samples to be analyzed. To applythe first measuring scheme as described above, the sensing window of oneof the output channels can be coated with a receptor layer such as anantibody layer using e.g. Protein A (measurement channel). A Protein Acoated sensor surface can be used to promote the binding and enhanceproper orientation of the receptor for further analyte binding.Furthermore, coating the sensor surface with Protein A results inreduction of non-specific binding to the sensor surface. An additional(reference) channel may be coated only with Protein A. The body fluidsample, e.g. serum containing a specific analyte, e.g. a biomarker, canbe applied (simultaneously) in both measurement and reference channels(see schematic in FIG. 2.A). Coating the sensor surface of the referencechannel with Protein A may also contribute to the reduction of thenon-specific binding, in this case of the serum components, in analogywith the measurement channel. Compared to the used measuring approach inwhich usually the sample is applied only in the measurement channel, andtherefore it is not possible to differentiate between the sensor signalcaused by the specific binding of the analyte to the receptor layerimmobilized on the sensor surface and the non-specific signal caused bythe binding of other components in the sample solution to the sensorsurface, this scheme provides the advantageous effect that thenon-specific binding occurring in the reference channel, which is alsoreduced by coating its sensor surface with Protein A in similar way asthe measurement channel, can largely compensate the non-specific bindingthat occurs (simultaneously) in the measurement channel. Therefore thedifferential signal between the measurement channel and the referencechannel is most probably caused by the specific binding of the analyteonto the antibody layer immobilized in the measurement channel,considering a comparable non-specific binding of other components thatare present in the sample in both measurement channel and referencechannel.

In a further application of this measuring scheme in the YI or MZI orother interferometer based sensor configurations, both measurementchannel and reference channel can be first coated with Protein A oranother protein or molecule that exhibits the similar functionality asProtein A, e.g. reduction of non-specific binding to the sensor surface,in this case in both the measurement channel and the reference channel,followed by coating with ‘clean’ serum sample (i.e. serum withoutspecific analyte to be measured) or other (post-)blockingagents/solutions, consisting of one or a combination (simultaneous orsequential) of reference fluid(s), which are used to block non-specificbinding sites. Commonly used blocking agents/solutions include, butwithout limitation, BSA (bovine serum albumin), serum, non-fat dry milk,casein, gelatin in PBS, etc. Next, the body fluid sample, e.g. serumcontaining specific analyte, can be applied in both channels (seeschematic in FIG. 2.B). In this configuration, because both measurementand reference channels were initially fully coated with non-specificcomponents being present in the ‘clean’ serum sample, addition of serumcontaining specific analyte may result mostly in sensor signal caused bythe binding of the specific analyte to the antibody layer immobilized inthe measurement channel, while additional non-specific signal caused bythe binding of other components in the sample is expected to benegligible or much lower than specific binding because the most of thenon-specific binding regions/sites are already occupied/blocked. Assuch, a lower non-specific signal, which is further compensated betweenthe measurement channel and the reference channel, may thereforecontribute in a more accurate signal corresponding to the specificbinding.

In a further measuring scheme, a second reference channel, e.g. in amultichannel YI based sensor such as schematically depicted in FIG. 3,or any other interferometer configuration having at least 3 sensingchannels, namely a measurement channel and two reference channels, whichis pre-coated e.g. with Protein A, may be further coated with ‘clean’serum sample (see schematic in FIG. 2.C). Coating with Protein A heremay have the same purpose as it may have in the case of the measurementchannel and reference channel, namely to reduce the non-specific bindingto the sensor surface. The differential signal that may result betweenthe reference channel and the second reference channel is largely due totemperature differences between these channels and other disturbingfactors, resulting in the so-called drift. A difference in thetemperature of the sample solutions, which are flowed in these channels,may also result in a temperature difference between them. Furthermore, atemperature difference can occur e.g. due to the binding event takingplace in the measurement channel where heat exchange with thesurrounding may occur. Because the signal due to the drift in themeasurement channel occurs simultaneously with the signal due to thespecific binding, during the time frame of a binding event it ispractically impossible to discriminate between the sensor signal due tospecific binding and the signal due to the drift. In this measuringscheme, the drift signal measured between the reference channel and thesecond reference channel may be used to correct/estimate the driftsignal that occurs simultaneously between the measurement channel andthe reference channel in addition to the specific signal in themeasurement channel. This could be achieved e.g. by determining therelation between the signals for each pair of channels prior toapplication of the sample solution containing the specific analyte. Bycorrecting/reducing the drift signal, this measuring scheme may resultin a further improvement of the accuracy of the signal measured for thespecific binding. This scheme could be possible if the interferencesignals between the measurement channel and two reference channels areacquired simultaneously or sequentially.

It is noted that in this embodiment, the sample—possibly containing theanalyte—is preferably not brought into contact with the second referencechannel, whereas this sample is preferably brought into contact with thefirst reference channel and with the measurement channel(s).

In a further measuring scheme, a third reference channel, e.g. in amultichannel YI based sensor, as schematically depicted in FIG. 3, whichis pre-coated e.g. with Protein A, may be further coated with ‘clean’serum sample (see schematic in FIG. 2.D). Coating with Protein A herehas the same purpose as in the case of the measurement channel,reference channel and second reference channel, namely to reduce thenon-specific binding to the sensor surface of these channels.

The differential signal that may result between the second referencechannel and the third reference channel is mostly due to temperaturedifferences between these channels and other disturbing factors,resulting in the so-called drift, whereas the differential signalbetween the reference channel and the second reference channel is due totemperature differences between these channels and other disturbingfactors resulting in drift signal as well as some non-specific bindingof the analyte at the sensor surface of the reference channel.

The differential signal that may result between the measurement channeland the reference channel is due to the specific binding of the analyteat the sensor surface of the measurement channel, drift signal betweenthe measurement channel and the reference channel as well as thenon-specific binding of the analyte at the sensor surface of thereference channel. Because the signal due to the drift between themeasurement channel and the reference channel occurs simultaneously withthe signal due to the specific binding in the measurement channel aswell as the non-specific binding of the analyte in the referencechannel, during the time frame of a binding event it is practicallyimpossible to discriminate between the sensor signal due to specificbinding in the measuring channel, the non-specific binding of theanalyte in the reference channel and the signal due to the drift betweenthe measurement channel and the reference channel. In this measuringscheme, the drift signal measured between the second reference channeland the third reference channel may be used to correct/estimate thedrift signal that occurs simultaneously between the reference channeland the second reference channel as well as the drift signal that occursbetween the measurement channel and the reference channel. This could beachieved e.g. by determining the relation between the signals for eachpair of channels prior to application of the sample solution containingthe specific analyte. By correcting/reducing the drift signal betweenthe reference channel and the second reference channel, the non-specificbinding of the analyte in the reference channel can be estimated.Furthermore, by correcting the drift signal between the measurementchannel and the reference channel and estimating the non-specificbinding of the analyte in the reference channel, this measuring schememay result in even a further improvement of the accuracy of the signalmeasured for the specific binding of the analyte in the measurementchannel. This scheme could be applied if the interference signalsbetween the measurement channel and 3 reference channels are acquired,e.g. simultaneously or sequentially.

It is noted that in this scheme the sample, potentially containing theanalyte to be detected, is preferably not brought into contact with thesecond and the third reference channel, whereas this sample willpreferably be exposed to the first reference channel and the measurementchannel(s).

In a similar fashion, the measuring schemes described above could beapplied in a MMI (multimode interference) based interferometric sensordevice with multiple sensing regions (ref. WO2010090514 andNL20092002491). In an MMI based sensor, light beam from a(monochromatic) light source, e.g. a laser, is coupled to an MMIcoupler, in which the multimode interference structure may be arrangedto allow propagation of different propagation modes. Along thepropagation path, at least a measurement region and a reference regionare provided (FIG. 5). Binding of analyte particles in the fluid with aspecific receptor such as antibody, which is provided along themeasurement region, can cause a change in propagation of at least one ofthe modes, and may provide for a change in the interference between themodes. As a result, a change in the light pattern as provided by thedifferent modes onto the detector, which e.g. may be positioned at theendface of the multimode structure, may occur, hence allowing to detecta propagation characteristic by an analysis of the pattern provided ontothe detector.

Each measurement region can be provided with a sensing window to allowapplication of fluid samples to be analyzed. To apply the firstmeasuring scheme as described above, the sensing window of one of thesensing regions can be coated with a receptor layer such as an antibodylayer using e.g. Protein A (measurement region). A Protein A coatedsensor surface can be used to promote the binding and enhance properorientation of the receptor for further analyte binding. Furthermore,coating the sensor surface with Protein A may result in reduction ofnon-specific binding to the sensor surface. An additional second (i.e. areference) region may be coated only with Protein A. The body fluidsample, e.g. serum containing a specific analyte, e.g. a biomarker, canbe applied (simultaneously) in both measurement and reference regions(see schematic in FIG. 2.A). Coating the sensor surface of the referenceregion with Protein A may also contribute to the reduction of thenon-specific binding, in this case of the serum components, in analogywith the measurement region. Compared to the used measuring approach inwhich usually the sample is applied only in the measurement region, andtherefore it is not possible to differentiate between the sensor signalcaused by the specific binding of the analyte to the receptor layerimmobilized on the sensor surface and the non-specific signal caused bythe binding of other components in the sample solution to the sensorsurface, this scheme provides the advantageous effect that thenon-specific binding occurring in the reference region, which is alsoreduced by coating its sensor surface with Protein A in similar way asthe measurement region, can largely compensate the non-specific bindingthat occurs simultaneously in the measurement region. Therefore thedifferential signal between the measurement region and the referenceregion is most probably caused by the specific binding of the analyteonto the antibody layer immobilized in the measurement region,considering a comparable non-specific binding of other components thatare present in the sample in both measurement region and referenceregion.

In a further application of this measuring scheme in the MMI basedinterferometric sensor, both measurement and reference regions can befirst coated with Protein A or another protein or molecule that exhibitsthe similar functionality as Protein A, e.g. reduction of non-specificbinding to the sensor surface, in this case in both the measurementregion and the reference region, then with ‘clean’ serum sample (serumwithout specific analyte to be measured) or other (post-)blockingagents/solutions, consisting of one or a combination (simultaneous orsequential) of reference fluid(s), which are used to block non-specificbinding sites. Commonly used blocking agents/solutions include, butwithout limitation, BSA (bovine serum albumin), serum, non-fat dry milk,casein, gelatin in PBS, etc. Next, the body fluid sample, e.g. serumcontaining specific analyte, can be applied in both regions (seeschematic in FIG. 2.B). In this configuration, because both measurementand reference regions were initially fully coated with non-specificcomponents being present in the ‘clean’ serum sample, addition of serumcontaining specific analyte may result mostly in sensor signal caused bythe binding of the specific analyte to the antibody layer immobilized inthe measurement region, while additional non-specific signal caused bythe binding of other components in the sample is expected to benegligible. As such, a lower non-specific signal, which is furthercompensated between the measurement region and the reference region, maytherefore contribute in a more accurate signal corresponding to thespecific binding.

In a further measuring scheme, a second reference region of the MMIbased interferometric sensor, which is pre-coated e.g. with Protein A,may be further coated with ‘clean’ serum sample (see schematic in FIG.2.C). Coating with Protein A here has the same purpose as in the case ofthe measurement region and reference region, namely to reduce thenon-specific binding to the sensor surface. The differential signal thatmay result between the reference region and the second reference regionis largely due to temperature differences between these regions andother disturbing factors, resulting in the so-called drift. A differencein the temperature of the sample solutions, which are flowed throughthese regions, may also result in a temperature difference between them.Furthermore, a temperature difference can occur e.g. due to the bindingevent taking place in the measurement region where heat exchange withthe surrounding may occur. Because the signal due to the drift in themeasurement region occurs simultaneously with the signal due to thespecific binding, during the time frame of a binding event it isimpossible to discriminate between the sensor signal due to specificbinding and the signal due to the drift. In this measuring scheme, thedrift signal measured between the reference region and the secondreference region may be used to correct/estimate the drift signal thatoccurs simultaneously between the measurement region and the referenceregion in addition to the specific signal in the measurement region.This could be achieved e.g. by determining the relation between thesignals for each pair of regions prior to application of the samplesolution containing the specific analyte. Correction/reduction of thedrift signal may result in a further improvement of the accuracy of thesignal measured for the specific binding. This scheme could be possibleif the interference signals between the measurement region and tworeference regions are acquired, e.g. simultaneously or sequentially.

In a further measuring scheme, a third reference region RRG 3 asschematically depicted in FIG. 5, which is pre-coated e.g. with ProteinA, may be further coated with ‘clean’ serum sample (see schematic inFIG. 2.D). Coating with Protein A here has the same purpose as in thecase of the measurement region, reference region and second referenceregion, namely to reduce the non-specific binding to the sensor surfaceof these regions.

The differential signal that may result between the second referenceregion RRG 2 and the third reference region RRG 3 is mostly due totemperature differences between these regions and other disturbingfactors, resulting in the so-called drift, whereas the differentialsignal between the reference region RRG and the second reference regionRRG 2 is due to temperature differences between these regions and otherdisturbing factors resulting in drift signal as well as somenon-specific binding of the analyte at the sensor surface of thereference region.

The differential signal that may result between the measurement regionMRG and the reference region RRG is due to the specific binding of theanalyte at the sensor surface of the measurement region, drift signalbetween the measurement region and the reference region as well as thenon-specific binding of the analyte at the sensor surface of thereference region. Because the signal due to the drift between themeasurement region and the reference region occurs simultaneously withthe signal due to the specific binding in the measurement region as wellas the non-specific binding of the analyte in the reference region,during the time frame of a binding event it is practically impossible todiscriminate between the sensor signal due to specific binding in themeasuring region, the non-specific binding of the analyte in thereference region and the signal due to the drift between the measurementregion and the reference region. In this measuring scheme, the driftsignal measured between the second reference region and the thirdreference region may be used to correct/estimate the drift signal thatoccurs simultaneously between the reference region and the secondreference region as well as the drift signal that occurs between themeasurement region and the reference region. This could be achieved e.g.by determining the relation between the signals for each pair of regionsprior to application of the sample solution containing the specificanalyte. By correcting/reducing the drift signal between the referenceregion and the second reference region, the non-specific binding of theanalyte in the reference region can be estimated. Furthermore, bycorrecting the drift signal between the measurement region and thereference region and estimating the non-specific binding of the analytein the reference region, this measuring scheme may result in even afurther improvement of the accuracy of the signal measured for thespecific binding of the analyte in the measurement region. This schemecould be applied if the interference signals between the measurementregion and three reference regions are acquired, e.g. simultaneously orsequentially.

In a (bio-)sensor, the binding between the receptor such as antibody andthe specific analyte is usually slow; it could take hours beforecomplete saturation of the binding curve occurs. However, by analyzingthe initial slope (˜ minutes) of the analyte binding curve, one canexactly determine the amount of analyte that has been present in thesample. As such one does not need to record the binding curve until itreaches full saturation in order to be able to determine how muchanalyte is present in the sample that is measured. In order to do sofirst one has to analyze for each receptor-analyte combination the slopeof the binding curve. Next, the exact amount of analyte needs to becorrelated to the slope of the binding curve such as to be able toexactly determine the quantity of the analyte that is present in thetest sample. Furthermore, the software used for the analysis of theinterference pattern must be tuned and pre-programmed for each set ofanalyte with its specific receptor that is at hand in the sensingwindow. In addition, the software can be adjusted to interpret the slopeof the binding curve for the binding of an analyte to its specificreceptor that is present in the sensing window.

Another advantage of all the schemes described above is that by applyingthe sample containing the specific analyte simultaneously in themeasurement and reference regions/channels, the bulk refractive indexsignal, which is caused when different sample solutions, which havedifferent refractive indices, are successively applied onto the sensorsurface, can be compensated between these regions/channels. As a result,the slope of the analyte binding curve obtained during the first fewminutes after the application of a sample onto the measurementregion/channel is largely caused by the binding of the specific analyteto the antibody layer immobilized on the sensor surface of themeasurement region/channel. Because the slope achieved during the firstfew minutes after a binding event is initiated is used to estimate thespecific analyte concentration based on a pre-determined calibrationcurve, which can be obtained by determining the sensor signal fordifferent specific analyte concentrations in the sample solution, thenthe compensation/reduction of the signal caused by the bulk refractiveindex may contribute to the further improvement of the accuracy of thesensor signal that is used for rapid estimation (˜ minutes) of thespecific analyte concentration.

In an alternative measuring scheme, sensor surface of a measurementregion is first coated with a receptor, which next to antibody, can beDNA string, enzyme, functional protein or other specifically analytebinding substance; later serum sample containing a specific analyte,e.g. a biomarker, is applied. Next, a dedicated solution, e.g. HClacidic or ionic gradient solution, may be flowed to remove preferablyonly the specific analyte particles, but not the serum components thatare non-specifically bound on the sensor surface. The signalchange/decrease that is measured with respect to a reference region cancorrespond to the amount of analyte particles that are detached from theantibody layer (see FIG. 6.A). In a further application of thismeasuring scheme, the reference region can be coated with ‘clean’ serumsample (serum that does not contain the specific analyte to bemeasured).

Next, both measurement region and reference region can be simultaneouslywashed with a dedicated solution, e.g. HCl acidic or ionic gradientsolution (see FIG. 6.B). The differential signal between these tworegions may result in a more accurate signal corresponding to the amountof analyte particles that were initially specifically bound to and laterdetached from the antibody layer on the measurement region because thepossible removal of the serum components from the measurement regionmight be compensated by simultaneous removal of the serum componentsfrom the reference region.

In analogy with the measuring scheme described above, as illustrated inFIG. 2.C, a second reference region may be coated with ‘clean’ serum(serum that does not contain the specific analyte to be measured) andwashed simultaneously with the measurement region and other referenceregion with a dedicated solution such as HCl acidic solution. Thedifferential signal between the reference region and the secondreference region, which is largely caused due to the temperature changesbetween these regions and other disturbing factors (drift), may befurther used to correct for the drift signal between the first(measurement) region and the reference region, hence it may improvefurther the accuracy of the signal corresponding to the specific bindingin the measurement region, in complete analogy with the measuring schemeillustrated in FIG. 2.C.

The measuring schemes illustrated in and described with reference toFIG. 6 may be combined with the measuring schemes illustrated in anddescribed with reference to FIG. 2. E.g. the measuring schemeillustrated in FIG. 2.C may be combined with measuring schemeillustrated in FIG. 6.C, as presented in FIG. 6.D, i.e. first sensorsurface of a measurement region is coated with a receptor layer followedby coating of the measurement region, a reference region and a secondreference region with ‘clean’ serum sample. Next, the sample containingthe analyte is applied in the measurement region and the referenceregion. The sensor signal measured between the measurement region andthe reference region is largely caused by the binding of the specificanalyte to the antibody layer immobilized in the measurement region,while additional non-specific signal caused by the binding of othercomponents in the sample is expected to be negligible or much lower thanspecific binding because most of the non-specific binding regions/sitesare already occupied/blocked during coating with “clean” serum sample.The drift signal measured between the reference region and the secondreference region may be used to correct/estimate the drift signal thatoccurs simultaneously between the measurement region and the referenceregion, which could be achieved e.g. by determining the relation betweenthe signals for each pair of regions prior to application of the samplesolution containing the specific analyte, potentially improving theaccuracy of the signal corresponding to the specific binding in themeasurement region.

Finally, the measurement region, the reference region and the secondreference region can be washed simultaneously with a dedicated solutionsuch as HCl acidic solution. The differential signal between themeasurement region and the reference region may correspond to the amountof analyte particles that were initially specifically bound to and laterdetached from the antibody layer in the measurement region because thepossible removal of the serum components from the measurement regionmight be compensated by simultaneous removal of the serum componentsfrom the reference region. The differential signal between the referenceregion and the second reference region, which is largely caused due tothe temperature changes between these regions and other disturbingfactors (drift), may be further used to correct for the drift signalbetween the measurement region and the reference region, hence it mayimprove further the accuracy of the signal corresponding to the specificbinding in the measurement region.

In this combined measuring scheme, more (accurate) information can beobtained about the sensor signal corresponding to the binding of thespecific analyte to the antibody layer immobilized in the measurementregion, potentially leading to a higher specificity and sensitivity.

The aforementioned measuring schemes may be further combined with theuse of multiple wavelengths and/or polarizations. For eachwavelength/polarization, all the measuring schemes as described above indetail can be applied in the same way. Using more than onewavelength/polarization, in addition to the improvement of the accuracyof the signal that corresponds to the specific binding, bycompensating/reducing the nonspecific binding contribution and otherdisturbing factors such as drift, the sensor signal that is measured forthe specific binding of an analyte to the receptor layer immobilizedonto the sensor surface of a measurement region/channel can be furtherimproved. This new measuring scheme may be particularly useful forspecific detection of relatively large analyte particles such asviruses, bacteria and cells in a complex medium such asbody/animal/plant fluid (serum, plasma, blood, sputum, etc.), milk,waste streams, etc. Use of multiple wavelengths and/or polarizationscould offer the possibility to better discriminate between the specificbinding of large analyte particles such as viruses, bacteria and cells,and non-specific binding of the components that are present in thecomplex medium, e.g. proteins, DNA molecules, etc. For instance, usingthree different wavelengths, e.g. 488, 568 and 647 nm, because of thedispersion phenomenon, three different phase change signals between ameasurement region/channel and a reference region/channel can bemeasured independently and (quasi-) simultaneously from each other.Consequently, a system of three independent equations can be obtainedbased on which three different contributions, e.g. specific binding oflarge analyte particles, non-specific binding of the other componentspresent in the complex medium and bulk refractive index can besimultaneously determined. Simultaneous detection of non-specificbinding of proteins and specific binding of viruses or bacteria ispossible due to the difference in sensitivity coefficients towardsproteins (˜10 nm), viruses (˜100 nm) and bacteria or cells (1000 nm) fordifferent wavelengths. By subtracting/reducing further the contributionof the non-specific binding when more than one wavelength/polarizationis used, the accuracy of the sensor signal measured for the specificbinding will be further improved, potentially leading to an even higherspecificity and sensitivity.

Furthermore, use of multiple wavelengths/polarizations could result inan increase of the signal-to-noise ratio (SNR) of the sensor signalbecause more information is achieved regarding binding events.

Use of additional wavelengths can allow estimation of other possiblecontributions, e.g. one of these contributions may be the temperaturechange that occurs during an immunoreaction or during a similar reactionwith e.g. DNA/RNA or another receptor.

Integration with Light Scattering & Imaging of the Interference in theChip

In addition to the application of measuring schemes as previouslydescribed and use of the multiple wavelengths/polarizations, lightscattering from the sensing regions/windows on top of the opticalwaveguide chip OPC, as schematically shown in FIG. 8, may besimultaneously acquired and used to provide additional informationregarding the specific binding events occurring in the sensingregions/channels in order to further improve the accuracy of the signalthat corresponds to the specific binding. This embodiment may beemployed in an MMI type interferometer configuration as well as in otherinterferometer configurations.

Upon binding of analyte particles on the sensing regions, the intensityof the scattered light from these regions will change, which furthercould give an indication about the amount of analyte particles bound onthe sensing regions. Furthermore, discrimination based on the size ofanalyte particles such as proteins, viruses or bacteria could bepossible because the scattering signal depends on the particle size andoptical properties such as refractive index. This information could beused e.g. to better discriminate between the specific binding of largeanalyte particles such as viruses, bacteria and cells, and non-specificbinding of proteins that could be present in a body fluid sample that isbeing analyzed, in addition to the discrimination/estimation that isachieved using previously measuring schemes and multiplewavelengths/polarizations. In order to detect the scattering, a mirrorMIR or other suitable optics, may be positioned above respectively underthe optical chip OPC, so as to direct at least a part of the scatteredlight onto (e.g. a part of) the detector CCD.

Furthermore, the intensity distribution of the interference pattern,e.g. between different excited modes in the multimode structure of a MMIbased sensor could be used as extra additional information to monitorbinding events occurring on the sensing regions on top of the MMImultimode structure. Upon binding of a specific analyte onto a givensensing region, the intensity distribution will be locally changed. Asthis change depends on the analyte concentration, imaging of theintensity distribution in the MMI multimode structure could allowon-line monitoring of this intensity change and consequently may enableestimation of the analyte concentration.

Combining the signals obtained from the analysis of the interferencepattern, light scattering from analyte particles and imaging of theinterference in the chip could provide more accurate information aboutthe specific analyte-receptor interactions by reducing/correctingnon-specific bindings and/or other disturbing factors such astemperature changes, which may lead to higher specificity, accuracy andsensitivity of the sensor. Also, this scheme may provide a higher SNR ofthe sensor signal because more information about the binding events isachieved.

In each one of the embodiments described in this document, next to orinstead of liquid samples, vapours and gas samples (e.g. air) could beanalyzed, e.g. when the gas is pre-treated, concentrated and dilutedinto a liquid, e.g. PBS buffer. This could be useful e.g. for detectionof airborne pathogenic micro-organisms such as viruses and bacteria inhospitals, emergency clinics, etc. A pre-concentration step may benecessary to increase the concentration in a given volume to detectablevalues as well as to obtain statistically relevant data. Apre-concentration step could also be applied to liquid samples whenlarge volumes need to be analyzed, e.g. water, beer, etc.

Gasses could also be detected by using gas absorbent layers that arespecific towards a given gas component, e.g. CO2, toxic gasses, etc.

Solid samples could also be analyzed when these samples arediluted/suspended into a liquid, e.g. PBS buffer.

The (bio-)sensor device comprises a (portable) measurement system PODand a lab-on-a-chip (LOC) system. The LOC, an embodiment of which beingdepicted in FIG. 10A, comprises an inlet INL, a fluid supply (in thisexample comprising a (micro-)fluidic cuvette FCV), a sensing part SRGcomprising the measurement and reference regions and in most instancescompleted by an outlet OTL for disposing the fluid or disposing air orother gas as a result of a supply of the sample into the sensing part.The (micro-)fluidic part may also in part or in full be comprised in theportable measurement system. The measurement region and/or referenceregion(s) can be pre-coated with specific receptor molecules, such asantibodies, DNA strings, enzymes, functional proteins or otherspecifically analyte binding substances in order to make the chipselective for one particular analyte. Pre-coating can be performed wellin advance of actual measurements and pre-coated chips can be packed andshipped, but pre-coating can also just precede the actual measurementwhereas the sensor device can offer the means (flowing fluids) to coatthe chip. Hence, a function of “chip loader” could also be accomplishedby the portable measurement system. The working principle of theportable (bio-) measurement system is schematically presented in FIG. 9:First the sample (highly schematically indicated by SAM in FIG. 9) to beanalyzed is delivered to the inlet of the lab-on-a-chip system. Certainsamples can be diluted, e.g. with buffer (that may be pre-packed withinthe chip), e.g. PBS, to improve the sample flow towards the sensingregions/windows of the (bio-)sensor (1). The sample will flow from the(LOC) inlet to the sensing regions/windows via (micro-)fluidic channels.This could be achieved e.g. by using a (micro-) fluidic channelconfiguration that provides capillary forces or using a micro-pump topush the fluid from the LOC inlet towards sensing regions.(Micro)-fluidic channel configuration, e.g. height, width, etc., couldbe varied such as to create different binding kinetics of variouscomponents (e.g. specific analytes and non-specific components that maybe present in the fluid sample solution) in the sensing regions/windows.The difference in binding kinetics between specific analytes andnon-specific components in (micro-)fluidic channels with differentconfigurations (which may be estimated for a given configuration) mayallow to further discriminate between the specific analyte binding andnon-specific binding of other components. Furthermore, the flow speedcould be varied, e.g. creating different flow speeds in different(micro-)fluidic channels, to allow an additional difference in bindingkinetics between specific analytes and non-specific components uponapplication of the fluid sample solution and/or the reference fluidand/or the blocking fluid or other fluid into the sensingregions/windows.

Upon insertion of the LOC system into the portable measurement system(2), a measurement will be (automatically) started and the analytebinding will be recorded. Analysis of the binding curve in the first fewminutes will provide the analyte concentration whereas the receptorlayer pre-coated into the chip yields information about the type ofanalyte detected. Examples of analytes include, but without limitation,biomarkers, DNA molecules, viruses, bacteria, cells, etc. (3). Next todiagnosis applications, this measuring scheme may be preferable forscreening purposes as well, e.g. in airports, emergency clinics orinfected areas, where a rapid response is especially important.

The lab-on-a-chip system may also be used for continuous samplemonitoring purposes. In this case the sample can be flowed over the LOCthrough the sensing regions/windows of the (bio-)sensor for a given timeperiod. This measuring scheme may be useful when a sample, which e.g. iscollected from a processing or production unit, has to be monitoredcontinuously, possibly in line, for the presence of certain analytes,e.g. pesticides in (drinking/waste) water, antibodies in milk or yeastin beer. A (continuous or not) pre-treatment (e.g. concentration,mixing, etc.) step may precede the actual measurement.

In a preferable configuration, the lab-on-a-chip system can be held by achip holder CPH, which e.g. could be made of a plastic material, e.g.Delrin, on top of which the optical waveguide chip resides. The latter(the optical waveguide chip) can be made of e.g. Silicon or othersuitable optical materials. The (micro-)fluidic part of the LOC can e.g.be made of PDMS (Polydimethylsiloxane), PMMA (Polymethylmethacrylaat) oranother biocompatible material. All these LOC parts can be integratedinto one chip system (see FIG. 10). Integration of the (micro-)fluidicpart into the LOC system can be preferable e.g. for minimization of thesample leakage that may occur when the sample is flowed through thesensing regions/windows of the optical chip. Minimization of the sampleleakage is further preferable to prevent contamination of the LOC systemand contamination of the portable measurement system in which the LOC isread-out, which may further result in an improvement of the operatorsafety. In this configuration, the size of the optical waveguide chipcan be kept as small as possible, which may contribute in minimizationof the costs per test, without deteriorating the sensing performancesuch as sensitivity and stability as well as the multiplexingcapability, which means that also in this miniaturized chip layout theremay be multiple measurement regions. In other words, one LOC can havemultiple sensing regions/windows and can thus detect simultaneously(various) multiple analytes (e.g. for panel testing purposes). Eachmeasurement region can be coupled to one or more reference regions, e.g.to allow application of the measurement schemes as described previously.Alternatively, one reference region can also be coupled to one or moremeasurement regions. Minimal costs are necessary in order to offer theLOC as a one-off disposable, but the LOC can also be designed such thatit can be re-usable (see below).

The holder may be designed such that, upon positioning of the(micro-)fluidic cuvette on top of the optical chip (see a schematicexample of each component of the LOC system and the integrated system inFIG. 10.B), the fluidic channels of the cuvette are properly alignedwith respect to the sensing regions/windows that are realized on theoptical chip. To do so, the holder can be etched or otherwise configuredin such a way that the (micro-)fluidic cuvette can be positioned on itas shown in FIG. 10.B. The optical chip can be positioned at the bottomof the etched structure of the (plastic) holder, e.g. by etching achannel that is slightly wider than the optical chip. In that way,lateral positioning of the optical chip can be obtained. The positioningalong the other direction, which may be less critical, can be arrangedwith respect to the endface of the (plastic) holder. After alignment ofthe optical chip at the bottom of the etched structure is achieved, the(micro-)fluidic cuvette can be inserted in this structure by pushing itdown from the top of the structure until it comes in contact with theoptical chip, aligning it with respect to the holder. Details on apossible fibre-to-chip coupling are provided below. Because the opticalchip is aligned with respect to the (plastic) holder and also the(micro-)fluidic cuvette is aligned with respect to the holder, then the(micro-)fluidic cuvette will be automatically aligned with respect tothe optical chip. As a result, the fluidic channels of the cuvette willbe aligned with respect to the sensing regions/windows of the opticalchip.

Once aligned with respect to the (plastic) holder, the optical chipcould also be permanently positioned on it using e.g. a bondingtechnique.

The lab-on-a-chip system can be built such that it may beinterchangeable. The fluidic connection with the LOC system may bearranged such as to allow a fast interchangeability, e.g. it may beconfigured as a modular unit, that can be quickly positioned uponinserting of the lab-on-a-chip system into the portable measurementsystem, in that way enabling performance of a rapid test measurement.This configuration may be preferable in combination with anauto-alignment method to enable a faster and better coupling of the(laser) light beam into the optical waveguide chip after insertion ofthe system into the portable measurement system. Furthermore, receptorlayers used to pre-coat the chip can be better preserved in such anintegrated, closed system. Such a closed system may protect thereceptors such as antibodies from (fast) deteriorating and may alsoprevent contamination of the sensing regions/windows after pre-coatingprocess and prior to application of analyte samples.

The interchangeable lab-on-a-chip system may be disposable, implying useof a new system for each sample that has to be measured, which may bepreferable e.g. for safety reasons, when a sample containing aninfectious pathogen such as virus needs to be analyzed. In a disposableLOC system, the sample that is first delivered to the LOC inlet can befurther flowed to the sensing regions/windows e.g. by using a(micro-)fluidic channel configuration that provides capillary forces.This configuration is preferable when a sample containing an infectiouspathogen needs to be analyzed because the part of the flow system thatis used to bring the sample from an external reservoir to the sensingregions/windows, e.g. tubes, connectors, etc., will not be contaminated.

The LOC system may also be re-usable, e.g. using a regenerationprocedure in which only the bound analyte particles are removed using adedicated solution, e.g. HCl acidic or ionic gradient solution, or whenboth the antibody layer and analyte are completely removed from thesensor surface using a given cleaning procedure, e.g. washing with astrong acidic solution such as 100% HNO₃.

In the measurement system, different components of the (optical) set-up,which are used to read-out the LOC system, such as the light source,e.g. a laser diode; incoupling optics, e.g. polarizer, lenses, feedbacksystem for automated light coupling into the optical chip, e.g. a piezosystem, and/or a fibre-to-chip coupling system; chip holder; fluidsupply, whether or not coupled to a (micro-)fluidic pump, to add fluidto the sensing regions/windows of specific chip channels; a detector,e.g. a CCD camera (including components used to obtain an optimaloutcoupling of the light from the optical chip to the CCD array chip,such as lenses, filters or matching oil that could be used when the CCDchip can be mounted onto the optical chip endface), single boardcomputer, touchscreen, electronic circuit and power supply can beintegrated into the measurement system (see FIG. 11). A computer board,which may be used for data collection and analysis, is an inexpensivesolution that can perform overall sensor device control. In anotherconfiguration, a personal digital assistant (PDA) may also be used toperform the device operation, which may result in a more compactmeasurement system e.g. having lower power consumption. The measurementsystem can be battery-operated to enable stand-alone operation. In theembodiment depicted in FIG. 11, a fluid supply system with pump andvalves may be provided as a separate module, whereby the (micro-)fluidicpart may be integrated on the chip or at least partially on or in themeasurement system. In this embodiment, each channel is individually andspecifically addressed.

This closed configuration of the measurement system may be preferable toprevent or reduce different disturbing factors such as background lightsources as well as temperature and humidity variations caused by theexternal environment. Furthermore, a compact, portable measurementsystem is potentially very useful for on-site field applications as wellas in remote or developing regions without easy access to sophisticatedlaboratory facilities. In this document, the terms measurement region,measurement channel, measurement window, sensing window, and sensingregion should be understood so as to refer to same or similar items.Similarly, the terms reference region, reference channel, and referencewindow should be understood so as to refer to the same or similar items.Also, the terms waveguide structure, planar structure, optical chip andlab-on-a-chip may be considered to refer to same or similar items.

It is remarked that next to the signal of the light scattered from theanalyte particles, the signal obtained from the specific labelling, e.g.fluorescent, magnetic, etc., of the analyte particles can be acquired insimilar fashion using a dedicated optical scheme. The signal due tospecific labelling could provide additional information about the sensorsignal corresponding to the specific binding of the analyte, henceimproving even further the accuracy and the sensitivity of the sensor.

Further, it is remarked that an antigen can also play the role of thereceptor to detect e.g. the presence of an antibody in a given samplesolution. This could be achieved by coating the sensor surface with anantigen layer and applying the sample solution containing the antibodyto the antigen-coated sensor surface.

Still further, it is remarked that the use of a white-lightsupercontinuum source may enable a high resolution discriminationbetween the sensor signal caused by the changes, e.g. in refractiveindex, etc, in the region within a few nanometres from the sensorsurface and the signal caused by the changes taking place in the regionbetween a few nanometres to e.g. hundred of nanometres from the sensorsurface. This could be preferable to obtain more accurate informationabout processes that may occur in close vicinity with the sensor surfacesuch as conformational changes in biomolecules, protein aggregations,etc.

Still even further, the disclosed method and the measurement system,next to detection of an analyte in a fluid sample, could also be usedfor quantitative measurement of affinities and kinetics of variousbiomolecular interactions such as protein-protein, protein-DNA,receptor-ligand, etc.

FIG. 12A depicts the sensor signal Ss (i.e. phase signal) on the y-axisversus time T on the x-axis, based on which an effect of embodimentsaccording to the invention will be illustrated. The sensor signalcomprises a differential signal that expresses a difference between themeasurement region and the reference region over time. Each depictedcurve represents a differential signal between the measurement channeland the reference channel. The measurement channel is provided with thereceptor. It is noted that the phase signal represents a phase of aspatial frequency peak in the fast fourier transform of the interferencepattern, as explained above.

The improvement of the sensor signal stability in an interferometricsensor when both the measurement channel and the reference channel arecoated with the blocking fluid, in accordance with an embodiment of theinvention, is illustrated in FIGS. 12A and B with data obtained fromexperimental measurements. In this figure, the differential signalmeasured between the measurement channel and the reference channelduring the measurement of a sample fluid, is presented. FIG. 12Apresents the measurement of the complete binding curve; FIG. 12B is aclose view of the sensor signal baseline before and immediately afterapplication of the sample fluid in the measurement channel. Curve Bindicates the differential signal measured between the measurementchannel and the reference channel when both the measurement channel andthe reference channel are coated with the blocking fluid. Curve Aindicates the differential signal measured between the measurementchannel and the reference channel when only the measurement channel isprovided with the blocking fluid. The sample is applied at Ta (forexample to replace blocking fluid, reference fluid or other fluid),washing is performed at Tw.

As it can be clearly seen from this figure, the differential signalmeasured when both the measurement channel and the reference channel areprovided with the blocking fluid (curve B) is much more stable comparedto the differential signal when only the measurement channel is providedwith the blocking fluid (curve A). Therefore, the differential signalcorresponding to the specific binding, which is determined by comparingthe signal baselines before application of the analyte sample at Ta andafter washing step at Tw, can be estimated with a higher accuracy whenboth the measurement channel and the reference channel are provided withthe blocking fluid, resulting further in a higher sensitivity. Thus, acharacteristic of the (reference) optical pattern obtained beforeapplication of the sample is compared with the characteristic of theoptical pattern obtained during and/or after providing of the sample.

In accordance with a further embodiment of the invention, the samplefluid is applied on both the measurement channel and the referencechannel after providing both channels with the Blocking Fluid; this isdepicted in curve C.

If the sample fluid will be provided not only into the measurementchannel, but also into the reference channel, which both were previouslycoated with the blocking fluid, then next to a more stable differentialsignal, as illustrated above, there may be also a reduction/compensationof the bulk effect (in FIG. 12A referred to as Delta 2 while an effectof the binding of the analyte is referred to as Delta 1) between thesample fluid and the blocking fluid. This may result in a more accurateestimation of the initial slope of the binding curve, which may be usedto derive the presence of the analyte during the very first minutesafter application of the sample fluid.

In FIG. 12A the differential signal measured between the measurementchannel and the reference channel when the sample fluid is provided toboth channels (after both channels were coated with blocking fluidbefore) is depicted as curve C

FIG. 12A depicts a measurement of a complete binding curve. As expected,a binding slope measured when the sample fluid is provided into both themeasurement channel and the reference channel (curve C) is corrected fora bulk effect between the blocking fluid and the sample fluid (curve B),which is present when the sample fluid is provided only into themeasurement channel.

Furthermore, when the sample fluid will be provided into both themeasurement channel and the reference channel, the additionalnon-specific binding that may be caused by sample fluid components otherthan specific analyte binding may be compensated between the measurementchannel and the reference channel, contributing therefore to a moreaccurate differential signal corresponding (almost) one-to-one to thespecific binding of the analyte of interest.

FIG. 12B depicts an enlarged, detailed view of an initial binding slopeSL when:

-   -   only the measurement channel is provided with the blocking fluid        and the sample fluid is applied only to the measurement channel        (curve A);    -   both the measurement channel and the reference channel are        provided with the blocking fluid and the sample fluid is applied        either only in the measurement channel (curve B) or in both the        measurement and the reference channel (curve C).

In this FIG. 12B, curve A has been shifted upwards along the y-axis(compared to FIG. 12A), for a better comparison with the two othercurves B and C.

When only the measurement channel is provided with the blocking fluidand the sample is applied only in the measurement channel (curve A),next to a bigger slope (compared to curve C), due to the bulk effectbetween the blocking fluid and the sample fluid (being also present incurve B), there is also an unstable signal, which e.g. can be caused bythe temperature drift, that deteriorates the accuracy of determining thebinding slope that corresponds to the specific binding, which furthercan result in a lower sensitivity.

Detection limit in accordance with the invention, as achieved so farexperimentally, is ˜1 fg/ml for an average mid-sized protein S100β,which considering a penetration depth of evanescent field of ˜200 nm, isequivalent to ˜10⁻³ fg/mm². It should be noted that the sensitivity ofthe method is still one order of magnitude above the noise level. Thismeans that when the signal to noise ratio is improved with imageanalysis algorithms, the sensitivity will be even higher.

1. A method for detecting an analyte in a fluid sample, comprising: a)providing a measurement region and a reference region, the measurementregion being provided with a receptor for binding the analyte; b)providing at least one light beam so as to travel along the measurementregion and along the reference region; c) providing the fluid sampleinto at least the measurement region; d) detecting by means of adetector an optical pattern provided by the at least one light beamafter having travelled along the measurement region and the referenceregion; and e) deriving a presence of the analyte in the fluid samplefrom the detected optical pattern, wherein prior to c) a blocking fluidis provided along the measurement region and along the reference region.2. The method according to claim 1, wherein the fluid sample is providedinto the measurement region and the reference region.
 3. The methodaccording to claim 1, comprising: detecting before c) by means of thedetector a reference optical pattern provided by the at least one lightbeam after having travelled along the measurement region and thereference region, wherein d) is performed at least once during or afterproviding of the fluid sample into at least the measurement region, andwherein e) comprises: comparing a characteristic of the referenceoptical pattern with the characteristic of the optical pattern detectedin d), and obtaining the presence of the analyte therefrom.
 4. Themethod according to claim 3, wherein the characteristic of the opticalpattern and the reference optical pattern comprises a phase of afrequency component in a spatial frequency spectrum of the opticalpattern, the frequency component from an interference between the atleast one light beam having travelled along the measurement region andhaving travelled along the reference region.
 5. The method according toclaim 1, wherein a second reference region is provided, wherein d)further comprises measuring a deviation between the reference region andthe second reference region, and wherein e) further comprises estimatinga disturbance from the deviation measured in d) between the referenceregion and the second reference region, and correcting the informationconcerning the presence of the analyte for the estimated disturbance. 6.The method according to claim 5, wherein c) further comprises providinga reference fluid at least along the second reference region.
 7. Themethod according to claim 5, wherein the disturbance comprises a driftbetween the measurement region and the reference region, wherein priorto c) a first drift is measured between the measurement region and thereference region and a second drift is measured between the referenceregion and the second reference region, wherein a drift relation isdetermined between the first drift and the second drift, and wherein thedrift between the measurement region and the reference region isestimated from the determined drift relation and the deviation asmeasured in d) between the reference region and the second referenceregion.
 8. The method according to claim 5, wherein a third referenceregion is provided, wherein d) further comprises measuring a deviationbetween the second reference region and the third reference region, andwherein e) further comprises estimating a further disturbance from thedeviation measured in d) between the second reference region and thethird reference region, and correcting the deviation between thereference region and the second reference region for the estimateddisturbance.
 9. The method according to claim 8, wherein the furtherdisturbance comprises an effect of non-specific binding.
 10. The methodaccording to claim 8, wherein a fourth reference region is provided,wherein d) further comprises measuring a deviation between the thirdreference region and the fourth reference region, and wherein e) furthercomprises estimating a still further disturbance from the deviationmeasured in d) between the third reference region and the fourthreference region, and correcting the deviation between the referenceregion and the second reference region and between the second referenceregion and the third reference region for the estimated disturbance. 11.The method according to claim 10, wherein the still further disturbancecomprises a bulk effect between the sample solution and the blockingand/or reference fluid.
 12. The method according claim 1, wherein e)comprises determining an initial slope of a measurement curve andderiving the presence of the analyte from the determined initial slope.13. The method according to claim 12, wherein the initial slope of themeasurement curve is compared to pre-determined calibration datarelating the initial slope to different analyte concentrations.
 14. Themethod according to claim 1, comprising the further steps of: removingat least part of the analyte from the receptor layer by a removalprocess, the optical pattern being detected before and after theremoval.
 15. The method according to claim 14, wherein a reference fluidis applied along the reference region and wherein the removal process isfurther performed along the reference region.
 16. The method accordingto claim 15, wherein the reference fluid is further applied along thesecond reference region, wherein the removal process is furtherperformed along the second reference region, and wherein e) comprisesderiving a drift between the measurement region and the reference regionfrom a drift measured between the reference region and the secondreference region, and correcting the information concerning the presenceof the analyte for the derived drift between the measurement region andthe reference region.
 17. The method according to claim 1, wherein thelight beam comprises at least two spectrally distinct wavelength ranges,the detection being performed for each of the wavelength ranges.
 18. Themethod according to claim 17, wherein three distinct wavelength rangesare comprised in the light beam, e) comprising determining analytebinding, non-specific binding and bulk refractive index from thedetected optical patterns for each of the wavelengths.
 19. The methodaccording to claim 1, wherein the light beam comprises a supercontinuumwavelength range, e) preferably comprising a monitoring processoccurring in close vicinity, with, preferably a nanometer distance froma sensor surface of at least the measurement region.
 20. The methodaccording to claim 1, wherein at least d) is repeated making use of adifferent state of polarization of the light beam, the detection beingperformed for each state of polarization.
 21. The method according toclaim 1, further comprising: detecting a scattering of light from themeasurement region and the reference region, and combining the detectedlight scattering with the detected optical pattern in order to derivethe presence of the analyte in e).
 22. The method according to claim 1,further comprising: detecting a spatial intensity distribution of thelight travelling through the measurement and reference regions, andcombining the detected local intensity distributions with the detectedoptical pattern, in order to derive the presence of the analyte in e).23. The method according to claim 1, wherein the measurement region andthe reference region are provided on or in a planar structure.
 24. Themethod according to claim 23, wherein the fluid sample and/or thereference fluid and/or the blocking fluid or other fluid is providedinto at least one of the measurement region and the reference region bya fluid supply, the method comprising holding the planar structure andthe fluid supply by a holder and aligning the fluid supply to at leastthe measurement region by the holder.
 25. The method according to claim1, wherein at least two measurement regions are provided, each beingprovided with a respective receptor for binding a respective analyte.26. A measurement system for detecting an analyte in a fluid sample,comprising: a measurement region and a reference region, the measurementregion being provided with a receptor for binding the analyte; a lightsource for generating at least one light beam a light guiding means forguiding the light beam along the measurement region and along thereference region; a fluid supply for providing the fluid sample and/orthe reference fluid and/or the blocking fluid or other fluid into themeasurement region and/or the reference region; a detector for detectingan optical pattern provided by the at least one light beam after havingtravelled along the measurement region and the reference region; and adata processing device for deriving a presence of the analyte in thefluid sample from the detected optical pattern.
 27. The measurementsystem according to claim 26, wherein at least the measurement regionand the reference region are provided on a chip structure, themeasurement system comprising a holder that holds the chip structure andthe fluid supply, the holder aligning the fluid supply to themeasurement and reference regions.
 28. A disposable measurementstructure comprising: a chip structure comprising a measurement regionand a reference region; a light guiding means for guiding a light beamalong the measurement and reference regions; a fluid supply for guidinga fluid sample into the measurement region and the reference region; anda holder for holding the chip structure and the fluid supply, the holderaligning the fluid supply to the measurement and reference regions.